Cardiopulmonary exercise testing in evaluation of patients of chronic obstructive pulmonary disease.

BACKGROUND: Objective assessment of severity in patients with chronic obstructive pulmonary disease (COPD) is mainly limited to pulmonary function testing performed at rest. But, accurate assessment of exercise capacity in patients with COPD may be possible with cardiopulmonary exercise testing (CPET). METHODS: Forty-three patients with stable COPD were included and were divided into three groups based upon the spirometry data as per the Global Initiative for Obstructive Lung Disease (GOLD) guidelines as follows: Group A: mild COPD, Group B: moderately severe COPD and Group C: severe COPD. Symptom-limited CPET was performed using treadmill on incremental continuous ramp protocol in all of them. RESULTS: Five patients (11.6%) had mild COPD; 16 (37.2%) had moderately severe COPD and the remaining 22 (51.6%) patients had severe COPD. Anaerobic threshold was attained in all the 43 patients. The dominant symptom at peak exercise were dyspnoea (n = 19) and both dyspnoea and leg fatigue (n = 7). The other causes of exercise limitation included dyspnoea with significant oxygen desaturation (n = 6); and dyspnoea with severe oxygen desaturation (n = 2). Six patients complained only of leg fatigue at peak exercise. A significant correlation between forced expiratory volume in the first second (FEV1) percent predicted and the predicted maximum oxygen uptake (VO2 max % predicted) was observed in all the three groups (r = 0.39, p = 0.011) but with marked variability of peak VO2 for a given degree of airflow obstruction. Twenty-three (53.5%) patients with low anaerobic threshold (< 30%) were identified as potential group likely to benefit from exercise training for pulmonary rehabilitation. CONCLUSIONS: Cardiopulmonary exercise testing is useful to determine the causes of exercise limitation and to assess the maximal exercise capacity of patients with COPD.

Zinc inhibits Bax and Bak activation and cytochrome c release induced by chemical inducers of apoptosis but not by death-receptor-initiated pathways.

Zinc has been known for many years to inhibit apoptosis but the mechanism remains unclear. Originally thought to inhibit an apoptotic endonuclease, zinc has subsequently been shown to inhibit steps earlier in the pathway. Since many additional steps in apoptosis have now been defined, we have re-evaluated the steps inhibited by zinc. In response to activation of the chemical-mediated death pathway by anisomycin, 0.3 mM zinc inhibited Bax and Bak activation, cytochrome c release, and all of the subsequent steps in apoptosis. In the receptor-mediated death pathway initiated by Fas or tumor necrosis factor, 3 mM zinc was required to inhibit apoptosis as judged by inhibition of caspase 3 activity and DNA digestion, but it failed to inhibit cytochrome c release, activation of Bax and Bak, or upstream signaling events in this pathway. These results are consistent with zinc selectively inhibiting activation of BH3-only proteins required in the chemical pathway but inhibiting downstream caspase activation in the death-receptor pathway.

Prevalence of coronary artery disease in patients with symptomatic peripheral vascular disease.

BACKGROUND: Prevalence of coronary artery disease has been reported to be quite high in patients with peripheral vascular disease in western literature. Therefore, it is important to study the coronary anatomy in patients with symptomatic peripheral vascular disease. METHODS AND RESULTS: Fifty-three patients presenting with symptoms of peripheral vascular disease underwent peripheral angiography in our institute during the last 2 years. The total number of vessels involved in these patients was 117. Fifteen patients had involvement of the upper limb vessels, 46 patients had involvement of the lower limb vessels and 6 patients had involvement of the carotid/vertebral arteries. Coronary arteriography was done in all the patients. Only 8 (15%) patients were found to have coronary artery disease with involvement of 11 arteries. Eighty-four (72%) peripheral vessels out of the 117 vessels involved showed total occlusion, whereas only 2 (18%) coronary arteries out of 11 vessels involved showed total occlusion. CONCLUSIONS: This study shows that the majority of patients with symptomatic peripheral vascular disease have normal coronaries, the extent of their involvement being low despite severe peripheral vascular disease.

Partial rescue of ethanol-induced neuronal apoptosis by growth factor activation of phosphoinositol-3-kinase.

BACKGROUND: Ethanol inhibition of insulin signaling pathways may contribute to impaired central nervous system (CNS) development in the fetal alcohol syndrome and brain atrophy associated with alcoholic neurodegeneration. Previous studies demonstrated ethanol inhibition of insulin-stimulated growth in PNET2 CNS-derived proliferative (immature) neuronal cells. We now provide evidence that the growth-inhibitory effect of ethanol in insulin-stimulated PNET2 cells is partly due to apoptosis. METHODS: Control and ethanol-treated PNET2 cells were stimulated with insulin and analyzed for viability, apoptosis, activation of pro-apoptosis and survival gene expression and signaling pathways, and evidence of caspase activation. RESULTS: Ethanol-treated PNET2 neuronal cells exhibited increased apoptosis mediated by increased levels of p53 and phospho-amino-terminal c-jun kinase (phospho-JNK), and reduced levels of Bcl-2, phosphoinositol 3-kinase (PI3 K), and intact (approximately 116 kD) poly (ADP ribose) polymerase (PARP), a deoxyribonucleic acid repair enzyme and important substrate for caspase 3. Partial rescue from ethanol-induced neuronal cell death was effected by culturing the cells in medium that contained 2% fetal calf serum instead of insulin, or insulin plus either insulin-like growth factor type 1 or nerve growth factor. The resulting enhanced viability was associated with reduced levels of p53 and phospho-JNK and increased levels of PI3 K and intact PARP. CONCLUSIONS: The findings suggest that ethanol-induced apoptosis of insulin-stimulated neuronal cells can be reduced by activating PI3 K and inhibiting pro-apoptosis gene expression and intracellular signaling through non-insulin-dependent pathways.

Aberrant expression of nitric oxide synthase III in Alzheimer's disease: relevance to cerebral vasculopathy and neurodegeneration.

Alzheimer's disease (AD) has heterogeneous pathology, in part due to the large subset of cases (AD+CVD) with superimposed vascular lesions that are sufficient in number and distribution to accelerate the clinical course of dementia. Brains with AD+CVD have lower densities of neurofibrillary tangles and A beta-amyloid diffuse plaques, and increased numbers of cerebral vessels exhibiting p53-associated apoptosis relative to brains with uncomplicated AD. AD and AD+CVD both exhibit altered expression of the nitric oxide synthase 3 (NOS-III) gene; however, in AD+CVD, reduced NOS-III expression in cerebral vessels is associated with an increased frequency of vascular lesions, vascular smooth muscle cell apoptosis, and A beta-amyloid plaques. In contrast, experimental and spontaneous focal acute and subacute cerebral infarcts are associated with increased NOS-III expression in perifocal neurons, glial cells, cerebrovascular smooth muscle and endothelial cells, and diffuse A beta-amyloid plaque formation. This suggests that ischemic injury and oxidative stress can precipitate NOS-III-mediated cell loss and neurodegeneration. A role for aging-associated impaired mitochondrial function as a contributing factor in AD and CVD is suggested by the reduced levels of mitochondrial protein observed in AD and AD+CVD cortical neurons and vascular smooth muscle and endothelial cells. The aggregate findings suggest that cell loss and neurodegeneration may be mediated by somewhat distinct but overlapping mechanisms in AD and AD+CVD.

Oxygen free radical injury is sufficient to cause some Alzheimer-type molecular abnormalities in human CNS neuronal cells.

Cell loss and neuritic/cytoskeletal lesions represent two of the major categories of dementia-associated structural abnormalities in Alzheimer's disease (AD). Cell loss is ultimately mediated by apoptosis and mitochondrial DNA damage due to enhanced sensitivity to oxidative stress, but the mechanism responsible for the neuritic/cytoskeletal lesions including the abnormal proliferation of cortical neurites is not known. This study examines the potential role of oxygen free radical injury as a factor contributing to both cell death and neuritic sprouting cascades in AD. PNET2 human neuronal cells were treated with H2O2 (8 micro M to 88 micro M) for 24 hours and then analyzed for viability, DNA damage, and pro-apoptosis, survival, and sprouting gene expression and signaling. H2O2-treatment resulted in dose-dependent increases in cell death due to genomic and mitochondrial DNA damage associated with increased levels of 8-OHdG and the p53 and CD95 pro-apoptosis genes, reduced levels of the Bcl-2 survival gene, activation of JNK and p38 stress kinases, and inhibition of PI3 kinase survival signaling. However, the H2O2-treated cells also manifested increased expression of growth and sprouting molecules, including GAP-43, nitric oxide synthase 3, neuronal thread protein (NTP; approximately 17 kD and approximately 21 kD forms), proliferating cell nuclear antigen, and phospho-Erk MAPK, and normal levels of the AD-associated approximately 41 kD NTP species, cyclin dependent kinase 5 (cdk-5), and phospho-tau. In addition, the H2O2-treated cells had increased levels of p25, the catalytically active and stable cleavage product of p35, which regulates cdk-5 activity. Previous studies demonstrated p25 accumulation in AD brains and p25-induced hyperphosphorylation of tau and neuronal apoptosis. The findings herein suggest that oxygen free radical injury in human CNS neuronal cells is sufficient to cause some but not all of the pro-death and pro-sprouting molecular abnormalities that occur in AD.

Low levels of arsenic trioxide stimulate proliferative signals in primary vascular cells without activating stress effector pathways.

Chronic human exposure to low levels of inorganic arsenic increases the incidence of vascular diseases and specific cancers. Exposure of endothelial cells to environmentally relevant concentrations of arsenic trioxide (arsenite) induces oxidant formation, activates the transcription factor NF-kappaB, and increases DNA synthesis (Barchowsky et al., Free Radic. Biol. Med. 21, 783-790, 1996). We show, in the current study, that arsenite induces concentration-dependent cell proliferation or death in primary porcine aortic endothelial cells. Low concentrations caused cell proliferation and were associated with increased superoxide and H(2)O(2) accumulation, cSrc activity, H(2)O(2)-dependent tyrosine phosphorylation, and NF-kappaB-dependent transcription. These concentrations were insufficient to activate MAP kinases. However, the MAP kinases, extracellular signal-regulated kinase and p38, were activated in response to levels of arsenite that caused cell death. These data suggest that arsenite-induced oxidant accumulation and subsequent activation of tyrosine phosphorylation represent a MAPK-independent pathway for phenotypic change and proliferation in vascular cells.

Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1.

Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.

Neuritic sprouting with aberrant expression of the nitric oxide synthase III gene in neurodegenerative diseases.

Neuronal loss, synaptic disconnection and neuritic sprouting correlate with dementia in Alzheimer's disease (AD). Nitric oxide (NO) is an important synaptic plasticity molecule generated by nitric oxide synthase (NOS) oxidation of a guanidino nitrogen of L-arginine. Experimentally, the NOS III gene is modulated with neuritic sprouting. In a previous study, NOS III expression was found to be abnormal in cortical neurons, white matter glial cells, and dystrophic neurites in AD and Down syndrome brains. The present study demonstrates the same abnormalities in neuronal and glial NOS III expression with massive proliferation of NOS III-immunoreactive neurites and glial cell processes in other neurodegenerative diseases including: diffuse Lewy body disease, Pick's disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, multiple system atrophy, and Parkinson's disease. However, each disease, including AD, was distinguished by the selective alterations in NOS III expression and sprouting in structures marred by neurodegeneration. Double label immunohistochemical staining studies demonstrated nitrotyrosine and NOS III co-localized in only rare neurons and neuritic sprouts, suggesting that peroxynitrite formation and nitration of growth cone proteins may not be important consequences of NOS III enzyme accumulation. The results suggest that aberrant NOS III expression and NOS III-associated neuritic sprouting in the CNS are major abnormalities common to several important neurodegenerative diseases.

P53- and CD95-associated apoptosis in neurodegenerative diseases.

Apoptosis is likely to be an important mechanism of cell loss in neurodegenerative diseases, but the signaling cascades activated before DNA fragmentation have not yet been determined. p53 or CD95 gene up-regulation precedes apoptosis in many cell types, and a potential role for these molecules in apoptosis of neurons and glial cells has already been demonstrated in Alzheimer's disease (AD). To determine whether apoptosis in other neurodegenerative diseases is mediated by similar mechanisms, p53 and CD95 expression were examined in postmortem central nervous system tissues from patients with diffuse Lewy body disease (DLBD), Pick's disease (PkD), progressive supranuclear palsy (PSP), multiple system atrophy (MSA), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Down's syndrome plus Alzheimer's disease (DN+AD). Quantitative immunoblot analysis demonstrated higher temporal lobe levels of p53 and CD95 proteins in DLBD, PkD, and DN+AD, and higher temporal lobe levels of CD95 only in MSA and PSP relative to PD and aged controls (for all, p < 0.01). In histologic sections, increased p53 immunoreactivity was localized in neuronal and glial cell nuclei, neuronal perikarya, and dystrophic neuritic and glial cell processes in the frontal (Area 1 1) and temporal (Area 21) lobes in DLBD, PkD, and DN+AD, the motor cortex and spinal ventral horns in ALS, and the striatum and midbrain in DLBD, MSA, PD, and PSP. Increased CD95 expression and nuclear DNA fragmentation were present in the same cell types and structures that manifested increased nuclear p53 immunoreactivity. The results suggest that p53- or CD95-associated apoptosis may be a common mechanism of cell loss in several important neurodegenerative diseases. In addition, the presence of abundant p53-immunoreactive neurites and glial cell processes appears to be a novel feature of neurodegeneration shared by these distinct diseases.

Simultaneous quantitation of plasma doxorubicin and prochlorperazine content by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed and tested for simultaneous extraction, elution and determination of doxorubicin and prochlorperazine content in human plasma samples. The procedure consists of extraction through a conditioned C18 solid-phase extraction cartridge, elution from a Spherisorb C8 reversed-phase column by an isocratic mobile phase (60% acetonitrile, 15% methanol and 25% buffer) followed by detection with electrochemical and fluorescence detectors. Recovery of doxorubicin and prochlorperazine from pooled human plasma samples (n=3) containing 100 ng/ml of the two drugs was 77.8+/-3.5% and 89.1+/-6.0%, respectively. The lower limits of quantitation for doxorubicin and prochlorperazine in plasma samples were 6.25 ng/ml and 10 ng/ml, respectively. A linear calibration curve was obtained for up to 2 microg/ml of doxorubicin and prochlorperazine. This combination method may be of particular value in clinical studies where phenothiazines such as prochlorperazine are used to enhance retention of doxorubicin in drug resistant tumor cells.